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Decoding long nanopore sequencing reads of natural DNA.

Laszlo AH, Derrington IM, Ross BC, Brinkerhoff H, Adey A, Nova IC, Craig JM, Langford KW, Samson JM, Daza R, Doering K, Shendure J, Gundlach JH - Nat. Biotechnol. (2014)

Bottom Line: As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers).This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome.This work provides a foundation for nanopore sequencing of long, natural DNA strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.

ABSTRACT
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

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A quadromer map predicts current levels for previously unmeasured DNA. (a) Current levels observed for all possible 4-nucleotide sequences (quadromers) measured in eight segments of a 256-nucleotide de Bruijn sequence. (b) The black trace shows a consensus based on 22 reads of phi X 174 DNA. This is compared to predicted current levels based on the de Bruijn quadromer values. Error bars are the variance of the measured quadromer values. We use a consensus to correct for insertion/deletion errors caused by the stochastic motion of the phi29 DNAP11. (c) Absolute current difference between quadromer map and measured consensus for the ~100 level sequence shown in panel b using the de Bruijn quadromer map (blue) and the revised quadromer map (red). In most instances, the revised map improves the predictive ability of our map. The correlation coefficient between measured values and the de Bruijn quadromer values is 0.9905 (95% confidence bounds [0.9859–0.9936]). The correlation coefficient between measured values and the revised quadromer values is 0.9938 (95% confidence bounds [0.9908–0.9958]).
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Figure 2: A quadromer map predicts current levels for previously unmeasured DNA. (a) Current levels observed for all possible 4-nucleotide sequences (quadromers) measured in eight segments of a 256-nucleotide de Bruijn sequence. (b) The black trace shows a consensus based on 22 reads of phi X 174 DNA. This is compared to predicted current levels based on the de Bruijn quadromer values. Error bars are the variance of the measured quadromer values. We use a consensus to correct for insertion/deletion errors caused by the stochastic motion of the phi29 DNAP11. (c) Absolute current difference between quadromer map and measured consensus for the ~100 level sequence shown in panel b using the de Bruijn quadromer map (blue) and the revised quadromer map (red). In most instances, the revised map improves the predictive ability of our map. The correlation coefficient between measured values and the de Bruijn quadromer values is 0.9905 (95% confidence bounds [0.9859–0.9936]). The correlation coefficient between measured values and the revised quadromer values is 0.9938 (95% confidence bounds [0.9908–0.9958]).

Mentions: We constructed a 256 nucleotide-long cyclical de Bruijn sequence18 containing all possible combinations of four nucleotides (Supplementary Table 1). We divided the de Bruijn sequence into eight separate strands (Supplementary Table 1) and synthesized these with appropriate modifications to facilitate insertion into the pore, to initiate proper polymerase function and to allow for ion current calibration11, 17, 19 (Fig. 1a). Phi29 DNAP–based control of translocation results in two reads from each DNA molecule: (i) `unzipping` wherein one strand of the DNA moves 5' to 3' through the pore as the polymerase is forced to unzip the complimentary strand, and (ii) `synthesis' wherein the DNA moves 3' to 5' after the primer enters the DNAP's active site and the DNAP begins synthesizing a second complimentary strand11. As the polymerase moves along the strand, one nucleotide at a time, the identity of the quadromer within the MspA pore shifts in lock-step (Fig 1b), resulting in discrete changes in the measured ion current (Fig. 1c). We performed nanopore sequencing of all eight strands, averaging signals observed across multiple molecules of each strand to estimate the current level of the 256 quadromers, i.e. a `quadromer map' (Fig. 2a, Supplementary Figs. 2–4, Supplementary Table 2).


Decoding long nanopore sequencing reads of natural DNA.

Laszlo AH, Derrington IM, Ross BC, Brinkerhoff H, Adey A, Nova IC, Craig JM, Langford KW, Samson JM, Daza R, Doering K, Shendure J, Gundlach JH - Nat. Biotechnol. (2014)

A quadromer map predicts current levels for previously unmeasured DNA. (a) Current levels observed for all possible 4-nucleotide sequences (quadromers) measured in eight segments of a 256-nucleotide de Bruijn sequence. (b) The black trace shows a consensus based on 22 reads of phi X 174 DNA. This is compared to predicted current levels based on the de Bruijn quadromer values. Error bars are the variance of the measured quadromer values. We use a consensus to correct for insertion/deletion errors caused by the stochastic motion of the phi29 DNAP11. (c) Absolute current difference between quadromer map and measured consensus for the ~100 level sequence shown in panel b using the de Bruijn quadromer map (blue) and the revised quadromer map (red). In most instances, the revised map improves the predictive ability of our map. The correlation coefficient between measured values and the de Bruijn quadromer values is 0.9905 (95% confidence bounds [0.9859–0.9936]). The correlation coefficient between measured values and the revised quadromer values is 0.9938 (95% confidence bounds [0.9908–0.9958]).
© Copyright Policy
Related In: Results  -  Collection

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Figure 2: A quadromer map predicts current levels for previously unmeasured DNA. (a) Current levels observed for all possible 4-nucleotide sequences (quadromers) measured in eight segments of a 256-nucleotide de Bruijn sequence. (b) The black trace shows a consensus based on 22 reads of phi X 174 DNA. This is compared to predicted current levels based on the de Bruijn quadromer values. Error bars are the variance of the measured quadromer values. We use a consensus to correct for insertion/deletion errors caused by the stochastic motion of the phi29 DNAP11. (c) Absolute current difference between quadromer map and measured consensus for the ~100 level sequence shown in panel b using the de Bruijn quadromer map (blue) and the revised quadromer map (red). In most instances, the revised map improves the predictive ability of our map. The correlation coefficient between measured values and the de Bruijn quadromer values is 0.9905 (95% confidence bounds [0.9859–0.9936]). The correlation coefficient between measured values and the revised quadromer values is 0.9938 (95% confidence bounds [0.9908–0.9958]).
Mentions: We constructed a 256 nucleotide-long cyclical de Bruijn sequence18 containing all possible combinations of four nucleotides (Supplementary Table 1). We divided the de Bruijn sequence into eight separate strands (Supplementary Table 1) and synthesized these with appropriate modifications to facilitate insertion into the pore, to initiate proper polymerase function and to allow for ion current calibration11, 17, 19 (Fig. 1a). Phi29 DNAP–based control of translocation results in two reads from each DNA molecule: (i) `unzipping` wherein one strand of the DNA moves 5' to 3' through the pore as the polymerase is forced to unzip the complimentary strand, and (ii) `synthesis' wherein the DNA moves 3' to 5' after the primer enters the DNAP's active site and the DNAP begins synthesizing a second complimentary strand11. As the polymerase moves along the strand, one nucleotide at a time, the identity of the quadromer within the MspA pore shifts in lock-step (Fig 1b), resulting in discrete changes in the measured ion current (Fig. 1c). We performed nanopore sequencing of all eight strands, averaging signals observed across multiple molecules of each strand to estimate the current level of the 256 quadromers, i.e. a `quadromer map' (Fig. 2a, Supplementary Figs. 2–4, Supplementary Table 2).

Bottom Line: As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers).This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome.This work provides a foundation for nanopore sequencing of long, natural DNA strands.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, University of Washington, Seattle, Washington, USA.

ABSTRACT
Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Show MeSH
Related in: MedlinePlus